Methods for assessing efficacy of chemotherapeutic agents

ABSTRACT

Methods are provided for accurately predicting efficacy of chemotherapeutic agents. Methods of the invention increase the positive predictive value of chemosensitivity assays by assessing both the ability of a chemotherapeutic to destroy cells and the genetic propensity of those cells for resistance. Results obtained using methods of the invention provide insight into the in vivo effectiveness of a therapeutic, and lead to more effective chemotherapeutic treatment.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/336,659 flied Jan. 12, 2003 now abandoned, which claims priority toU.S. Application Ser. No. 60/417,439, filed Oct. 12, 2002. The entiredisclosures of the prior applications are incorporated by referenceherein.

FIELD OF THE INVENTION

The invention relates to methods for assessing efficacy ofchemotherapeutic agents.

BACKGROUND

Cancer chemotherapy involves the use of cytotoxic drugs to destroyunwanted cells in patients. Treatment may consist of using one or morecytotoxic drugs, depending on the nature of the disease being treated.However, drug toxicity and drug resistance are significant barrierseffective chemotherapy.

Toxicity from chemotherapeutic agents produces side effects ranging frommild trauma to death. Moreover, repeated exposure to chemotherapeuticdrugs is itself often fatal. As chemotherapeutic drugs are carried inthe blood, they are taken up by proliferating cells, including normalcells. Tissues with high growth rates such as bone marrow and epithelialtissues, including the gastrointestinal tract, are normally mostsusceptible to toxic side effects. Some drugs have additional toxiceffects on other tissues, such as the urinary tract, myocardium, orpancreas. Chemotherapeutic agents may cause direct injury to the heart,either acutely, in the form of myocardial tissue injury or dysrhythmias,or in a delayed or chronic fashion associated with congestive heartfailure.

Target cells, such as malignant or diseased cells, may be intrinsicallyresistant to chemotherapeutic drugs or they may acquire resistance as aresult of exposure. A target cell may be genetically predisposed toresistance to particular chemotherapeutics. Alternatively, the cell maynot have receptors or activating enzymes for the drug or may not bereliant on the biochemical process with which the drug interferes.Additionally, individuals may be inherently resistant to a drug due toaltered disposition of the drug in organs other than the tumor. Thesemechanisms include, but are not limited to, rapid metabolism to inactivespecies, failure to metabolize to an active species of drug, and rapidclearance or sequestration. Many of these aspects are encodedgenetically by normal polymorphisms in metabolic genes that actprimarily, but not exclusively, in the liver and gastrointestinal tractand the kidneys.

Acquired resistance also may develop after cells have been exposed to adrug or to similar classes of drugs. One example of acquired drugresistance is the multiple drug resistance phenotype. Multiple drugresistance is a phenomenon of cross-resistance of cells to a variety ofchemotherapeutic agents which are not structurally or functionallyrelated. This phenomenon is typically mediated by p-glycoprotein, a cellmembrane pump that is present normally on the surface of some epithelialcells. The protein actively removes drug from the cell, making itresistant to drugs that are substrates for the cell membrane pump.

A critical issue in cancer chemotherapy is the ability to select drugsthat not only affect cancer cell phenotype in cell culture assays, butare also not subject to resistance whether in the tumor or intrinsic tothe patient. The present invention addresses that issue.

SUMMARY OF THE INVENTION

The invention provides methods for accurately predicting efficacy ofchemotherapeutic agents. Methods of the invention increase the positivepredictive value of chemosensitivity assays by assessing both theability of a chemotherapeutic to affect tumor cells phenotype and thegenetic propensity of the patient for resistance to thechemotherapeutic. Results obtained using methods of the inventionprovide insight into the in vivo effectiveness of a therapeutic, andlead to more effective, individualized, chemotherapeutic choices.

According to the invention, a phenotype assay screens a therapeuticcandidate for the ability to affect the phenotype of tumor cells inculture. A therapeutic candidate that produces the desired phenotypiceffect (e.g., cell death, decreased motility, changes in cellularadhesion, angiogenesis, or gene expression, among others) then isscreened against genetic properties of cells of the patient which makeresistance to the therapeutic candidate likely or possible. Atherapeutic candidate that has a desired phenotypic effect on patienttumor cells and that does not appear to be subject to genetic-basedresistance is selected for use. As a result of combining phenotypic andgenetic data, use of the invention increases the likelihood that atherapeutic candidate, chosen on the basis of its ability to affectcellular phenotype, will be effective when administered to patients.

Accordingly, the invention provides methods for assessing efficacy ofchemotherapeutic agents comprising exposing cells to a chemotherapeuticagent, conducting an assay to determine whether the chemotherapeuticagent affects tumor cell phenotype, and identifying geneticcharacteristics of cells of the patient (which may or may not be tumorcells) that indicate a propensity for resistance to the chemotherapeuticagent.

In a preferred embodiment, a phenotypic assay for use in the inventioncomprises obtaining a tumor explant from a patient, culturing portionsof the explant, growing a monolayer of relevant cells from the explant,exposing the monolayer to a drug candidate, and assessing the ability ofthe drug candidate to alter tumor cell phenotype. A preferred phenotypicassay is disclosed in U.S. Pat. No. 5,728,541, and in co-owned,co-pending U.S. application Ser. No. 10/208,480, both of which areincorporated by reference herein.

Genotype analysis according to the invention is accomplished by anyknown method. A preferred method comprises comparing the genotype, orportion thereof, of cells obtained from the patient with genotypes knownto be associated with drug resistance generally, or specifically withrespect to a therapeutic candidate being evaluated. For example, theexistence in patient cells of a polymorphic variant that is known orsuspected to confer resistance to a therapeutic candidate would screenthat candidate out as a potential therapeutic against those cells.Genetic characteristics of patient cells are determined by methods knownin the art (e.g., sequencing, polymorphisms) as set forth below. Theimpact of a patient's genotype upon drug resistance may be determined byreference to genetic databases or libraries that catalog known mutationsor polymorphisms related to resistance.

The present invention also provides methods for selecting achemotherapeutic agent for treating a patient based on results obtainedfrom the phenotypic and genotypic assays. In a preferred embodiment, thepresent invention allows for the assessment of whether achemotherapeutic agent will be effective in treating a cancer whenadministered to a patient. According to the invention, chemotherapeuticagents or combinations of chemotherapeutic agents are selected fortreatment where an effect on cellular phenotype is observed andcharacteristics of genetic-based resistance are not observed.

Methods of the invention are useful in drug or chemotherapeutic agentscreening to provide information indicative of the in vivo reactivity ofthe cells, and thus the specific efficacy as to a particular patient.Methods of the invention are also useful to screen new drug candidatesfor therapeutic efficacy and to provide a basis for categorizing drugswith respect to the tumor types against which they will work best.

A phenotypic assay according to the invention is conducted on cellsobtained from a tumor explant from a patient. Genotypic assays of theinvention are performed on genetic data obtained from patient cells,regardless of their source. Thus, a genotypic assay can be performed onsomatic cells obtained from the patient or on cells from the same tumorthat is evaluated in the phenotypic assay. Assays of the invention canbe performed on an individualized basis or on a pool of samples obtainedfrom multiple individual patients. If assays are conducted on pooledsamples, the phenotypic characteristics of the pool of samples aredetermined followed by individualized genotypic assays on specificpatients. This allows multiplexing of the phenotypic portion of theassay.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides methods for assessing efficacy ofchemotherapeutic agents. Specifically, the invention provides methodsfor assessing the efficacy of chemotherapeutic agents based onphenotypic changes observed in tumor cells obtained from a patient andgenetic characteristics of the patient that indicate general or specificchemotherapeutic resistance. In one aspect of the invention, efficacy ofa chemotherapeutic agent is assessed based upon the results of thephenotypic and genotypic assays. In another aspect of the invention,chemotherapeutic agents are selected for treating a patient based on theresults of the phenotypic and genotypic assays.

The present invention is also useful for screening of therapeutic agentsagainst other diseases, including but not limited to, hyperproliferativediseases, such as psoriasis. In addition, the screening of agents thatretard cell growth (anti-cancer, anti-proliferative), including agentsthat enhance or subdue intracellular biochemical functions, areevaluated using methods of the present invention. For example, theeffects of therapeutics on the enzymatic processes, neurotransmitters,and biochemical pathways are screened using methods of the invention.Methods of the invention can be practiced on any type of cell obtainedfrom a patient, including, but not limited to, normal somatic cells,malignant cells, abnormal proliferating cells, and other diseased cells.Cells are obtained from any patient sample, including, but not limitedto, tumors, blood samples and buccal smears. The skilled artisanrecognizes that methods of the invention can be practiced using avariety of different samples.

In one step of the invention, a phenotype assay is employed to assesssensitivity and resistance to chemotherapeutic agents. The phenotypicassay is performed in vitro using cultured cells. The phenotype assayallows for identification and separation of target cells from othercells found in a tissue sample, as well as direct measurement andmonitoring of target cells in response to chemotherapeutic treatment.Direct measurements and monitoring of live cells are performed usingknown methods in the art including, for example, the measuring ofdoubling rate, fraction proliferative assays, monitoring of cytostasis,cell death, cell adhesion, gene expression, angiogenesis, cell motility,and others. Direct measurements also include known assays, such as thosedirected to measurement and monitoring of apoptosis, senescence, andnecrosis.

In another step of the invention, a genotype assay is performed todetermine whether cells from a patient comprise a genetic characteristicassociated with resistance to the chemotherapeutic agents. Genotypeassays reveal latent resistance to chemotherapeutic agents not observedby phenotypic assays. Genotypic assays may measure characteristics, suchas metabolism, toxic effects, absorption of a therapeutic candidate.

In one embodiment of the invention, the phenotypic assay is performedusing cell culture monolayers prepared from tumor cells. In a preferredembodiment, monolayers are cultured from cohesive multicellularparticulates generated from a tumor biopsy. Explants of tumor tissuesample are prepared non-enzymatically, for initial tissue culturemonolayer preparation. The multicellular tissue explant is removed fromthe culture growth medium at a predetermined time to both allow for thegrowth of target cells and prevent substantial growth of non-targetcells such as fibroblasts or stromal cells.

By way of example, in one embodiment of the invention, a cell culturemonolayer is prepared in accordance with the invention using thefollowing procedure. A biopsy of non-necrotic, non-contaminated tissueis obtained from a patient by any suitable biopsy or surgical procedureknown in the art. In a preferred embodiment, the tissue sample is tumortissue. The size of the biopsy sample is not central to the methodsprovided herein, but a sample is preferably about 5 to 500 mg, and morepreferably about 100 mg. Biopsy sample preparation generally proceedsunder sterile conditions. Cohesive multicellular particulates (explants)are prepared from the tissue sample by enzymatic digestion or mechanicalfragmentation. Ideally, mechanical fragmentation of the explant occursin a medium substantially free of enzymes that are capable of digestingthe explant. For example, the tissue sample may be minced with sterilescissors to prepare the explants. In a particularly preferredembodiment, the tissue sample is systematically minced by using twosterile scalpels in a scissor-like motion, or mechanically equivalentmanual or automated opposing incisor blades. This cross-cutting motioncreates smooth cut edges on the resulting tissue multicellularparticulates. After the tissue sample has been minced, the particles areplated in culture flasks (for example, 9 explants per T-25 flask or 20particulates per T-75 flask). The explants are preferably evenlydistributed across the bottom surface of the flask, followed by initialinversion for about 10-15 minutes. The flask is then placed in anon-inverted position in a 37° C. CO₂ incubator for about 5-10 minutes.In another embodiment in which the tissue sample comprises brain cells,the flasks are placed in a 35° C., non-CO₂ incubator. Flasks are checkedregularly for growth and contamination.

The multicellular explant is removed from the cell culture at apredetermined time, as described below. Over a period of a few weeks amonolayer is produced. With respect to the culturing of tumor cells, itis believed (without any intention of being bound by the theory) thattumor cells grow out from the multicellular explant prior tocontaminating stromal cells. Therefore, by initially maintaining thetissue cells within the explant and removing the explant at apredetermined time, growth of the tumor cells (as opposed to stromalcells) into a monolayer is facilitated. The use of the above procedureto form a cell culture monolayer maximizes the growth of tumor cellsfrom the tissue sample, and thus optimizes the phenotypic and genotypicassays.

Once a primary culture and its derived secondary monolayer tissueculture has been initiated, the growth of the cells is monitored tooversee growth of the monolayer and ascertain the time to initiate thephenotypic assay. Prior to the phenotypic assay, monitoring of thegrowth of cells may be conducted by visual monitoring of the flasks on aperiodic basis, without killing or staining the cells and withoutremoving any cells from the culture flask. Data from periodic countingor measuring is then used to determine growth rates or cell motility,respectively.

Phenotypic assays are performed on cultured cells using achemotherapeutic drug response assay with clinically relevant doseconcentrations and exposure times. One embodiment of the presentinvention contemplates a phenotypic assay that assesses whetherchemotherapeutic agents effect cell growth. Monolayer growth rate ismonitored using, for example, a phase-contrast inverted microscope. Inone embodiment, culture flasks are incubated in a (5% CO₂) incubator atabout 37° C. The flask is placed under the phase-contrast invertedmicroscope, and ten fields (areas on a grid inherent to the flask) areexamined using a 10× objective. In general, the ten fields should benon-contiguous, or significantly removed from one another, so that theten fields are a representative sampling of the whole flask. Percentagecell occupancy for each field examined is noted, and averaging of thesepercentages then provides an estimate of overall percent confluency inthe cell culture. When patient samples have been divided between twomore flasks, an average cell count for the total patient sample shouldbe calculated. The calculated average percent confluency should beentered into a process log to enable compilation of data—and plotting ofgrowth curves—over time. Alternatively, confluency is judgedindependently for each flask. Monolayer cultures may be photographed todocument cell morphology and culture growth patterns. The applicableformula is:

${{Percent}\mspace{14mu}{confluency}} = \frac{{estimate}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{area}\mspace{14mu}{occupied}\mspace{14mu}{by}\mspace{14mu}{cells}}{{total}\mspace{14mu}{area}\mspace{14mu}{in}\mspace{14mu}{an}\mspace{14mu}{observed}\mspace{14mu}{field}}$As an example, therefore, if the estimate of area occupied by the cellsis 30% and the total area of the field is 100%, percent confluency is30/100, or 30%.

Following initial culturing of the multicellular tissue explant, thetissue explant is removed from the growth medium at a predeterminedtime. In one embodiment, the explant is removed from the growth mediumprior to the emergence of a substantial number of stromal cells from theexplant. Alternatively, the explant may be removed according to thepercent confluency of the cell culture. In one embodiment of theinvention, the explant is removed at about 10 to about 50 percentconfluency. In a preferred embodiment of the invention, the explant isremoved at about 15 to about 25 percent confluency. In a particularlypreferred embodiment, the explant is removed at about 20 percentconfluency. By removing the explant in either of the above manners, acell culture monolayer predominantly composed of target cells (e.g.,tumor cells) is produced. In turn, a substantial number of non-targetcells, such as fibroblasts or other stromal cells, fail to grow withinthe culture. Ultimately, this method of culturing a multicellular tissueexplant and subsequently removing the explant at a predetermined timeallows for increased efficiency in both the preparation of cell culturesand subsequent phenotypic and genotypic assays for assessing efficacy ofchemotherapeutic agents.

In another embodiment, a phenotypic assay assesses whetherchemotherapeutic agents effect cell motility. Methods for measuring cellmotility are known by persons skilled in the art. Generally, thesemethods monitor and record the changes in cell position over time.Examples of such methods include, but are not limited to, videomicroscopy, optical motility scanning (for example, see U.S. Pat. No.6,238,874, the disclosure of which is incorporated by reference herein)and impedance assays. In a preferred embodiment, cell motility assaysare carried out using monolayer cultures of malignant cells as describedherein.

Cell culture methods of the invention permit the expansion of apopulation of proliferating cells in a mixed population of abnormalproliferating cells and other (normal) cells. The mixed population ofcells typically is a biopsy or sample from a solid tumor. A tissuesample from the patient is harvested, cultured and analyzed for geneticindicia of resistance to chemotherapeutics. Subcultures of the cellsproduced by the culture methods described above may be separatelyexposed to a plurality of treatments and/or therapeutic agents for thepurpose of objectively identifying the best treatment for the patient.By way of example, procedures for culturing the malignant cells anddetermining a phenotypic to a chemotherapeutic agent may be performed inthe following manner. First, a specimen is finely minced and tumorfragments are plated into tissue culture. The cells are then exposed togrowth medium, such as a tumor-type defined media with serum. The cellsare trypsinized, preferably, but not necessarily, when greater than150,000 cells grown out from tumor fragment. The cells are preferablyplated into a Terasaki plate at 350 cells per well. The cells areanalyzed to verify that a majority of cells are tumor epithelial cells.Non-adherent cells are removed from the wells. The cells are treatedwith 6 concentrations and 2 control lanes of chemotherapeutic agent oragents for preferably 2 to 4 hours. The chemotherapeutic agents areremoved by washing. The cells are incubated for preferably 3 days. Theliving cells are counted to calculate the kill dose that reduces by 40%the number of cells per well from control wells.

The culture techniques of the present invention result in a monolayer ofcells that express cellular markers, secreted factors and tumor antigensin a manner representative of their expression in vivo. Specific methodinnovations such as tissue sample preparation techniques render thismethod practically, as well as theoretically, useful.

According to the present invention, cells from a patient are analyzedfor genetic characteristics (abnormalities) specific to a patient.Genetic characteristic of a cell or cell population can be analyzedalone or in combination with other characteristics. Geneticcharacteristics of the invention can be, without limitation, a geneticpolymorphism or a mutation, such as an insertion, inversion, deletion,or substitution. In one embodiment, nucleic acids are isolated fromcells of a patient and analyzed to identify genotypic characteristics ofthe cells. The isolated nucleic acid is DNA or RNA. The nucleic acid,preferably, is analyzed in a microarray for DNA-encoded polymorphisms inthe coding or control regions of the gene. In another embodiment, thenucleic acid is analyzed in a microarray for aberrant expression of oneor more genes. In this embodiment, the microarray contains nucleic acidsthat are characteristic of known malignancies, as well as nucleic acids,that are not correlated with known malignancies so that previouslyunknown relationships between gene expression and a proliferativedisease or condition may be identified.

A preferred method of the invention comprises comparing the genotype, orportion thereof, of cells from a patient with genotypes known to beassociated with drug resistance generally, or specifically with respectto a therapeutic candidate being evaluated. For example, the existencein patient cells of a polymorphic variant that is known or suspected toconfer resistance to a therapeutic candidate would screen that candidateout as a potential therapeutic against those cells.

Methods for isolating and analyzing nucleic acids derived from the cellsare known in the art. The presence of known proliferation markers, suchas the aberrant expression of one or more genes, the epidermal growthfactor receptor (EGFR) cyclin D1, p16cyclin-kinase inhibitor,retinoblastoma (Rb), transforming Growth Factor β (TGFβ) receptor/smad,MDM2 or p53 genes, may be determined by, for example, northern blottingor quantitative polymerase chain reaction (PCR) methods (i.e., RT-PCR).

In one embodiment of the present invention, mRNA (polyA⁺ mRNA) isisolated and labeled cDNA is prepared therefrom. The labeled cDNA isprepared by synthesizing a first strand cDNA using an oligo-dT primer,reverse transcriptase and labeled deoxynucleotides, such as, Cy5-dUTP,commercially available from Amersham Pharmacia Biotech. Radio-labelednucleotides also can be used to prepare cDNA probes. The labeled cDNA ishybridized to the microarray under sufficiently stringent conditions toensure specificity of hybridization of the labeled cDNA to the arrayDNA.

In another embodiment of the invention, the labeled array is visualized.Visualization of the array may be conducted in a variety of ways. Forinstance, when the reading of the microarray is automated and thelabeled DNA is labeled with a fluorescent nucleotide, the intensity offluorescence for each discreet DNA of the microarray can be measuredautomatically by a robotic device that includes a light source capableof inducing fluorescence of the labeled cDNA and a spectrophotometer forreading the intensity of the fluorescence for each discreet location inthe microarray. The intensity of the fluorescence for each DNA sample inthe microarray typically is directly proportional to the quantity of thecorresponding species of mRNA in the cells from which the mRNA isisolated. It is possible to label cDNA from two cell types (i.e., normaland diseased proliferating cells) and hybridize equivalent amounts ofboth probe populations to a single microarray to identify differences inRNA expression for both normal and diseased proliferating cells. Toolsfor automating preparation and analysis of microarray assays, such asrobotic microarrayers and readers, are available commercially fromcompanies such as Gene Logic and Nanogen and are under development bythe NHGRI. The automation of the microarray analytical process isdesirable and, for all practical purposes necessary, due to the hugenumber and small size of discreet sites on the microarray that must beanalyzed.

In a further embodiment, DNA microarrays are used in combination withthe cell culturing method of the present invention due to the increasedsensitivity of mRNA quantification protocols when a substantially purepopulation of cells are used. For their ease of use and their ability togenerate large amounts of data, microarrays are preferred, whenpracticable. However, certain other or additional qualitative assays maybe preferred in order to identify certain markers.

In another embodiment, the presence of, or absence of, specific RNA orDNA species are identified by PCR procedures. Known geneticpolymorphisms, translocations, or insertions (i.e., retroviralinsertions or the insertion of mobile elements, such as transposons)often can be identified by conducting PCR reactions with DNA isolatedfrom cells cultured by the methods of the present invention. Where thesequence anomalies are located in exons, the genetic polymorphisms maybe identified by conducting a PCR reaction using a cDNA template.Aberrant splicing of RNA precursors also may be identified by conductinga PCR reaction using a cDNA template. An expressed translocated sequencemay be identified in a microarray assay, such as the Affymetrix p53assay.

In one embodiment, small or single nucleotide substitutions areidentified by the direct sequencing of a given gene by the use ofgene-specific oligonucleotides as sequencing primers. In a furtherembodiment, single nucleotide mutations are identified through the useof allelic discrimination molecular beacon probes, such as thosedescribed in Tyagi, S. and Kromer, F. R. (1996) Nature Biotech.14:303-308 and in Tyagi, S. et al., (1998) Nature Biotech. 16:49-53, thedisclosures of each of which are incorporated by reference herein.

Genotypic analysis may be based on experimentation or experience.Sources for such empirical data made be obtained from, but not limitedto clinical records and/or animal tumor transplant studies. Geneticcharacteristics found in the patient cells can be compared to a databasecontaining known tumor genotypes and their respective resistance tochemotherapeutic agents. In a preferred embodiment, a databasecontaining genotypes and their respective drug resistance profile isused to compare genotypic characteristics of the target cells toresistance to chemotherapeutic agents in vivo. Computer algorithms areuseful for carrying out pattern matching routines in complex systems,such as genetic data-mining. A linear regression algorithm, for example,can be utilized to analyze a database and identify the genotype mostclosely matching the genetic characteristics in the patient cells. Inone embodiment, a comparative analysis of genotypes is performed using aknown linear regression algorithm.

According to the invention, genotypic characteristics of patient cellsare analyzed to establish whether such characteristics are associatedwith resistance to chemotherapeutic agents in vivo. While theabove-mentioned genotypic assays are useful in the analysis of nucleicacids derived from cells produced by the culture methods embodied in thepresent invention, numerous additional methods are known in the generalfields of molecular biology and molecular diagnostics that may be usedin place of the above-referenced methods. Information obtained fromgenotypic assays is analyzed to determine efficacy of chemotherapeuticagents.

In a further embodiment of the invention, data obtained by practicingthe methods of the invention, including phenotypic, genotypic andpatient outcome information, is stored in databases. The contents ofthese databases include, but are not limited to, observed in vitrophenotypes (disease factors) and genotypes (host factors). By applyinganalytical techniques to the stored information, predictions ofchemotherapeutic efficacy can be made. Methods of the invention allowfor the skilled practitioner to accurately select an effective course ofchemotherapy for a patients, thus reducing the risk of treatment-relatedtrauma and resistance.

In one aspect of the invention, a course of chemotherapy is selectedbased on results obtained from the phenotypic and genotypic assays. Thepresent invention allows for the assessment of the likelihood of whetherchemotherapeutic agents will be effective in treating a malignancy in apatient. A phenotypic assay in combination with a genotypic assayoperates to minimize the risk of administering to a patient achemotherapeutic agent or combinations of chemotherapeutic agents towhich the tumor is resistant. In one aspect of the invention,chemotherapeutic agents or combinations of chemotherapeutic agents areselected for treatment where an effect on cellular phenotype is observedand the genotypic characteristics associated with resistance are notobserved.

Chemotherapeutic agents that effect cellular phenotype are potentialcandidates for use in the patient. Known procedures that screen forchemotherapeutic agents are time-consuming and expensive. In oneembodiment of the invention, chemotherapeutic agents that effectcellular phenotype and lack genetic changes associated with drugresistance are administered to the patient. In a further embodiment,genotypic characteristics observed in the genetic assay undergo acomparative analysis to determine if such characteristics are associatedwith drug resistance. In another embodiment, the phenotypic andgenotypic assays are performed in succession, thereby narrowing thescope of the genotypic comparative analysis, and reducing labor costsand associated expenses. In one aspect of the invention, when it isdetermined that a chemotherapeutic agent effects cellular phenotype andis not associated with resistance to cells having the genotypic change,a patient is treated with the chemotherapeutic agent.

The following examples provide further details of methods according tothe invention. For purposes of exemplification, the following examplesprovide details of the use of methods of the present invention in cancertreatment. Accordingly, while exemplified in the following manner, theinvention is not so limited and the skilled artisan will appreciate itswide range of application upon consideration thereof.

Example 1

A patient was diagnosed with breast cancer and chemotherapeutictreatment was prescribed by the treating physician. A tumor biopsy ofapproximately 100 mg of non-necrotic, non-contaminated tissue washarvested from the patient by surgical biopsy and transferred to alaboratory in a standard shipping container. Biopsy sample preparationproceeded as follows. Reagent grade ethanol was used to wipe down thesurface of a Laminar flow hood. The tumor was then removed, understerile conditions, from its shipping container, and cut into quarterswith a sterile scalpel. Using sterile forceps, each undivided tissuequarter was then placed in 3 ml sterile growth medium (Standard F-10medium containing 17% calf serum and a standard amount of Penicillin andStreptomycin) and minced by using two sterile scalpels in a scissor-likemotion. After each tumor quarter was minced, the particles were platedin culture flasks using sterile pasteur pipettes (9 explants per T-25 or20 particulates per T-75 flask). Each flask was then labeled with thepatient's code and the date of explantation. The explants were evenlydistributed across the bottom surface of the flask, with initialinverted incubation in a 37° C. incubator for 5-10 minutes, followed byaddition of about 5-10 ml sterile growth medium and further incubationin the normal, non-inverted position. Flasks were placed in a 35° C.,non-CO₂ incubator. Flasks were checked daily for growth andcontamination as the explants grew out into a cell monolayer.

Following initiation of prime cell culture of the tumor specimen, cellswere removed from the monolayers grown in the flasks for centrifugationinto standard size cell pellets. Each cell pellet was then suspended in5 ml of the above-described medium and was mixed in a conical tube witha vortex for 6 to 10 seconds, followed by manual rocking back and forth10 times. A 36 ml droplet from the center of each tube was then pipettedinto one well of a 96-well microtiter plate together with an equalamount of trypan blue, plus stirring. The resulting admixture was thendivided between two hemocytometer quadrants for examination using astandard light microscope. Cells were counted in two out of fourhemocytometer quadrants, under 10× magnification—only those cells whichdid not take up the trypan blue dye were counted. This process wasrepeated for the second counting chamber. An average cell count perchamber was calculated, and the optimum concentration of cells in themedium was determined.

Accommodating the above calculations, additional cell aliquots from the4 monolayers were separately suspended in growth medium via vortex androcking and were loaded into a Terasaki dispenser adapted to a 60-wellplate. Aliquots of the prepared cell suspension were delivered into themicrotiter plates using Terasaki dispenser techniques. Cells were platedinto 60-well microtiter plates at a concentration of 100 cells per well.

Twenty-four hours post-plating, the chemotherapeutic agent paclitaxelsold under the trademark TAXOL (Bristol-Myers Squibb Company) wasapplied to the wells in the microtiter plates. Three treatment rows inthe plates (Rows 2, 3, and 4) were designed to have escalatingpaclitaxel doses (1.0, 5.0, and 25 μM). Row 5 served as a control. Thepaclitaxel exposure time was two hours. The cells were allowed toincubate for another 72 hours so that inhibition of cell proliferationcan be observed. During this period, the growth inhibiting effect ofpaclitaxel was monitored by observing the percent of confluency of thecells. For each microtiter well, the percent of confluency of culturedcells was plotted as a function of time.

Since paclitaxel affected growth rate of the cultured cells, cells fromthe patient were subjected to genotypic analysis. DNA was isolated fromcells of the patient and analyzed for single nucleotide geneticpolymorphisms. Known genetic polymorphisms were identified in the DNA byconducting PCR reactions and sequencing or SNP detection byhybridizations of a region of interest in the DNA. The DNA region ofinterest from the patient cells was compared to corresponding regionsfrom known genetic banks and libraries (for example, GENBANK).

The phenotypic and genotypic assays were used in combination todetermine that paclitaxel was an efficacious course of treatment for thepatient. As a result, paclitaxel was administered to the patient.

Example 2

A patient was diagnosed with lung cancer and chemotherapeutic treatmentwas prescribed by the treating physician. A tumor biopsy ofapproximately 100 mg of non-necrotic, non-contaminated tissue washarvested from the patient by surgical biopsy and transferred to alaboratory in a standard shipping container. The biopsy sample wasprepared as described in Example 1. Twenty-four hours post-plating, thechemotherapeutic agent carboplatin sold under the trademark PARAPLATIN(Bristol-Myers Squibb Company) was applied to the wells in themicrotiter plates. The first three treatment rows in the plates (Rows 2,3, and 4) were designed to have escalating carboplatin doses (50, 200,and 1000 μM). Row 5 serves as a control. The carboplatin exposure timewas two hours. The cells were allowed to incubate for another 72 hoursso that inhibition of cell motility can be observed.

Cell motility was measured by calculating the distance a cell travelsover time. Cells were monitored using a digital video-camera mounted ona phase-contrast light microscope. To maintain the growth medium at 35°C., the microscope was fitted with a heated slide stage. After thecultured cells were incubated with carboplatin, cell migration wasrecorded under appropriate magnification (usually between 40× and 200×).During this period, the motility inhibiting effect of carboplatin wasdocumented by plotting the distance cells travel as a function of time.The distance cells travel was a determined using digital imagingtechniques known in the art.

Since carboplatin affected cell motility in the tumor cells, the cellswere subjected to genotypic analysis by comparing DNA from the culturedcells to known genetic banks and libraries. Known genetic polymorphismswere identified in the cultured cells by conducting PCR reactions andsequencing a region of interest in DNA isolated from the cultured cells.The DNA region of interest from the cultured cells was compared tocorresponding regions from known genetic banks and libraries (forexample, GENBANK).

Genetic characteristics observed in the genotypic assay were compared toa database of genetic characteristics that were known to be associatedwith resistance to carboplatin. The phenotypic and genotypic assays wereused in combination to determine that carboplatin was an efficaciouscourse of treatment for the patient. As a result, carboplatin wasadministered to the patient.

While the invention has been shown and described with reference tospecific preferred embodiments, it should be understood by those skilledin the art that various changes in form and detail may be made thereinwithout departing from the spirit and scope of the invention as definedby the following claims.

1. A method for assessing chemotherapeutic efficacy for a cancerpatient, comprising: allowing a multicellular tumor tissue explant ofthe patient to form a cell culture monolayer, removing the explant fromthe culture when the monolayer is at about 10 to about 50 percentconfluency and growing the monolayer, thereby preventing substantialgrowth of stromal cells; optionally preparing a subculture from themonolayer cells; conducting at least one phenotypic assay on cells fromthe monolayer or the subculture, so as to determine a tumor cellphenotype predictive of a drug's efficacy against the tumor; conductingat least one genotypic analysis of cells from the patient to determinethe presence or absence of a genotype predictive of resistance to saiddrug; thereby assessing whether the drug will be an effectivechemotherapy for the patient.
 2. The method of claim 1, wherein theexplant is from a solid tumor.
 3. The method of claim 1, wherein theexplant is prepared by mechanical fragmentation of tumor tissue in amedium substantially free of enzymes that are capable of digesting theexplant.
 4. The method of claim 1, wherein the explant is prepared bysystematic mincing of the tumor tissue.
 5. The method of claim 1,wherein the multicellular tissue explant is removed from the culturewhen the monolayer is at from 15% to 30% confluency.
 6. The method ofclaim 1, wherein the at least one phenotypic assay comprises a drugresponse assay.
 7. The method of claim 6, wherein the drug is ananti-proliferative agent.
 8. The method of claim 6, wherein the drugresponse assay measures one or more of cell death, cell growth,apoptosis, changes in cell motility, changes in cell adhesion, orchanges in gene expression upon exposure of the cultured cells to adrug.
 9. The method of claim 1, wherein the at least one phenotypicassay comprises an assay for expression of a cellular marker, secretedfactor, or tumor antigen.
 10. The method of claim 1, wherein the atleast one phenotypic assay comprises an assay for aberrant geneexpression of one or more proliferation markers.
 11. The method of claim10, wherein the one or more proliferation markers comprise at least onemarker selected from epidermal growth factor receptor (EGFR), cyclin D1,p16/cyclin-dependent kinase inhibitor, retinoblastoma (Rb), TransformingGrowth Factor β (TGFβ) receptor, MDM2, and p53.
 12. The method of claim1, wherein the genotype is an allelic variant and/or a single nucleotidepolymorphism.
 13. The method of claim 1, wherein the genotypic analysisis conducted on somatic cells from the patient.
 14. The method of claim1, wherein the genotypic analysis is conducted on a blood sample or abuccal smear from the patient.
 15. The method of claim 1, wherein thegenotypic analysis is conducted on cells of the monolayer or thesubculture.
 16. The method of claim 1, wherein the genotypic analysiscomprises determining a drug resistance profile for the patient.
 17. Themethod of claim 1, wherein the genotypic analysis is predictive of oneor more of drug metabolism, drug toxicity, and drug absorption.